1. Field of the Invention
The present invention relates to a capillary electrophoresis apparatus and, more particularly, to a capillary electrophoresis apparatus for reading an electrophoresis pattern obtained by subjecting nucleic acids or proteins to gel electrophoresis through a capillary with a high degree of sensitivity or resolution without requiring any special or expensive device such as a laser beam source.
2. Description of the Related Art
Heretofore, techniques for analysis by means of gel electrophoresis methods have been extensively utilized for the fragmentation and for the analysis of structures of protein acids and nucleic acids present as a polymer in a body of an animal or a plant. In many cases the gel electrophoresis method is utilized for experiments in which a sample can be procured beforehand in a very limited amount. Therefore, the analysis using the gel electrophoresis method requires a very high sensitivity to the detection of the sample.
In conventional techniques, a sample to be analyzed is first labelled with a radioactive isotope, the labelled sample is injected into a gel, and the resulting gel material is subjected to electrophoresis. After electrophoresis, the gel is attached to an X-ray film or the like for exposure to X rays to be emitted from the radioactive isotope labelled in the sample. After exposure of the gel material containing the labelled sample to the X-ray film or the like for a given period of time, the X-ray film is then developed, and the exposed pattern resulting from the radioactive isotope transfered onto the X-ray film is read as a gel electrophoresis pattern in order to analyze the structure of the proteins or nucleic acids of the sample.
The radioactive isotopes, however, are so hazardous that they should be handled and managed with extreme care and with high security. Recently, technologies for handling laser light and technologies of laser light sources, optical sensors and signal processing have greatly developed leading to the development of fluorescence detecting methods for detecting an electrophoresis pattern without requiring the use of such hazardous radioactive isotopes. The fluorescence detecting methods comprise labelling a sample with a fluorescent substance, subjecting the sample to electrophoresis, exciting the labelled fluorescent substance directly with a laser light source, and detecting the resulting fluorescent pattern, thereby resulting in the detection of the electrophoresis pattern.
As an example of the method for reading an electrophoresis pattern by the fluorescence detecting method, which has initially been developed, there is known a method for determining the sequence of a DNA as disclosed in Japanese Patent Unexamined Publication No. 61-173,158. An outline of a fluorescence detecting method utilized for the DNA sequence determining method will be described with reference to FIGS. 4 and 5 which show a schematic representation of the method for the detection of a gel electrophoresis pattern by the fluorescence detecting method. FIG. 4 is a block diagram showing an outline of a gel electrophoresis apparatus and FIG. 5 shows the details of a portion of a fluorescence detecting section of the gel electrophoresis apparatus of FIG. 4.
First, a description will be made of the device structure of the gel electrophoresis apparatus with reference to FIG. 4. The gel electrophoresis apparatus comprises a gel 31 in which electrophoresis is performed, a fine tube 32 for retaining the gel 31, an upper buffer solution container 33 and a lower buffer solution container 34, between which electrical field is applied to the gel 31 held in the fine tube 32, a first electrode 35a, a second electrode 35b, a light source 36 for exciting a fluorescent substance labelled in the electrophoresed sample, a detector 37 for detecting the fluorescence emitted from the sample, a data processing section 38 for processing electrical signals transmitted from the detector 37 and converting the fluorescent signals into electrical signals, and an electric power supply 39 for applying electrophoresis electrical field between the first and second electrodes 35a and 35b.
Next, an operation of the gel electrophoresis apparatus will be described by example where a DNA sample is electrophoresed as an object of electrophoresis and an electrophoresis pattern of the DNA sample is read with the gel electrophoresis apparatus. The DNA sample is first labelled with a fluorescent substance and the labelled sample is introduced into the gel filled in the fine tube 32 from the upper buffer solution container 33, and electrophoresis voltage of from several kV to approximately 10 kV is applied from the electric power supply 39 between the first and second electrodes 35a and 35b. As the DNA has negative charges, they migrate toward the positive second electrode 35b upon application of the electrophoresis voltage and they eventually reach the position of the light source 36. Thereafter the fluorescent substance labelled in the DNA sample is excited in this position upon exposure to laser beams emitted from the light source 36, thereby emitting fluorescence. The fluorescence is received by the detector 37 to be converted into electrical signals. The electrical signals representing the fluorescence are transmitted to the data processing section 38. The data processing section 38 determines the sequences of the fragments separated by their molecular weights (electrophoresis pattern).
In this case, the detector 37 for receiving the fluorescence emitted from the sample in the gel 31 operates as follows. Namely, as shown in FIG. 5 which is a partial transverse sectional view (as looked down upon from above), when the sample migrates in the gel 31 and the sample reaches the position of laser beams 40 emitted from the light source 36, the fluorescent substance labelled in the DNAs of the sample in the gel 31 is excited with the laser beams 40, thereby resulting in the emission of fluorescence 41 that in turn is received by the detector 37. The detector 37 fetches the received fluorescence 41 in a photomultiplier and converts the fluorescence into electrical signals and transmits the electrical signals to the data processing unit 38. The data processing unit 38 is arranged such that the sequences of the fragments in the sample are determined by the molecular weights on the basis of the peak positions of the intensity of the fluorescence 41.
When they are employed as a sample, DNA fragments are labelled with the fluorescent substance so as to have different fluorescent wavelengths corresponding to their ingredients, and to determine the DNA sequences of the four bases simultaneously by only one fine tube. The fluorescent substance which can emit four different fluorescent wavelengths, may include, for example, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TMRITC), and substituted rhodamine isothiocyanate (XRITC), respectively. Further, the gel electrophoresis apparatus of this type has a sensitivity to detection of DNAs in the order of 1.times.10.sup.-16 mole as high as the method using radioactive isotopes, when argon ion laser having wavelength of 488 nm or 514 nm is employed.
In addition, the above example briefly alludes to an example in which luminescence can be caused by taking advantage of chemical reaction energy. However, the example does not specifically disclose any procedures of the elution from a gel material, a reaction with a luminous substance, the removal of an unnecessary labelling substance, and the like. Actually, there are many specific problems to be solved in embodying techniques, which may include, for example, procedures for the supply of a source of chemically luminescent material, the mixture of the luminous substance with a labelling substance, and the removal of the labelling substance after luminescence in order to prevent background noises from being caused by residual substances.
The electrophoresis methods can be applied to, for example, the diagnosis of hereditary diseases, the investigation of DNAs in determining suspects of crimes and the investigation of the relationship between a parent and a child, in addition to the determination of the DNA sequence. In the diagnosis of hereditary diseases, it is currently possible to distinguish even one base from samples on the basis of the difference between the electrophoresis patterns by taking advantage of the difference in the structure under specific conditions (e.g. temperature or pH as causing a minute difference in denatured states of DNA) by the substitution of the base or bases inherent in each hereditary disease, such as single strand conformation polymorphism. The investigation of DNA in, for example, determining a suspect of crime and a parent-child relationship is made by comparing the difference in electrophoresis distances by taking advantage of a deviation in DNAs (polymorphism) between individuals.
In such experiments, the base length of a DNA is approximately 1,000 bases or less in many cases, and the gel employed for electrophoresis is polyacrylamide gel. In the case where the base length is several thousands, agarose gel is employed. Further, a gel electrophoresis apparatus of a flat plate type is employed for the comparison of the electrophoresis pattern of a sample with a reference DNA electrophoresis pattern. With such an gel electrophoresis apparatus, the sample and the reference DNA sample are subjected to gel electrophoresis side by side for a ready reference to the difference between the two electrophoresis patterns.
These methods, however, require taking care in a number of ways including, for example, sustaining homogeneity of a gel with high stability and maintaining the uniformity of temperature on the electrophoresis plates during electrophoresis processes. In particular, a strict management of temperature using a thermostat is required in single strand conformation polymorphism. The strict management of temperature increases the cost of a device expensive and makes its size large because the flat plate electrophoresis apparatus consumes a large amount of power and the amount of heat evolved is great. With respect to this point, the management can be performed easier by an electrophoresis apparatus of a capillary type because such apparatus can make its electrophoresis section smaller in size.
However, the prior art electrophoresis apparatus for reading the electrophoresis pattern obtained by the fluorescence detecting method of conventional technology requires the use of a laser light source of a unique type corresponding to the wavelength at which the fluorescent substance is to be excited. The conventional electrophoresis apparatus suffers from various disadvantages. One disadvantage is that the cost of the laser light source, which amounts for the total cost of the apparatus, is so great that the cost of the apparatus itself becomes expensive as well. Further, laser light emitted from the laser light source has a high energy density even if it would scatter, so that there is the risk of causing disorders or abnormality of vision, such as dyschromatopsia or blindness, if the laser light were to enter the naked eye. Hence, such a laser light source should be handled with great care and should be incorporated in the electrophoresis apparatus with great attention paid to security from such laser light. This also leads to making the electrophoresis apparatus expensive.